Redox biology is a rapidly expanding area of research. Recent studies have shown that the major cellular processes are regulated by redox, yet specific mechanisnns are poorly understood. With the exception of several exannples, the molecular targets of redox control, specificity of redox regulation, and the sets of proteins that maintain cellular redox homeostasis are not known. Thiol-based redox regulation has emerged as a prevalent mechanism to regulate cellular processes. Its major components, thioredoxin (Trx) and thioredoxin reductase (TR), are present in nearly all organisms. TRs control the redox state of Trxs, which in turn regulate cellular processes by controlling the redox state of cysteine (Cys) residues in proteins. Mammals have three TRs, and all three contain catalytic selenocysteine (Sec) residues; therefore, the mammalian Trx system is dependent on dietary selenium. Besides TRs and Trxs, numerous Trx-like proteins and other thiol oxidoreductases (i.e., proteins that use catalytic redox Cys) exist, but their functions are mostly unknown. It is also unknown which proteins are targeted by these enzymes. We would like to characterize TRs, Trxs, thiol oxidoreductases and protein targets regulated by these proteins to define the major pathways of thiol-based redox regulation in mammals. Specifically, we will carry out the following studies: (1) Characterize organismal sets of thiol oxidoreductases and their protein targets using bioinformatics, proteomics, and biochemical methods; (2) Analyze general features of thiol-based redox regulation; (3) Characterize interplay between cysteine and selenocysteine in the active sites of oxidoreductases; and (4) Examine interrelationship between hydrogen peroxide metabolism and thiol-based redox control. RELEVANCE (See instructions): Thioredoxin reductase and thioredoxin are the main redox regulators of cysteines in proteins, whereby controlling cellular processes. This system has been implicated in cell signaling, apoptosis, cancer development, and many other physiological processes. We propose to characterize functions of thioredoxin reductases, thiol oxidoreductases and their targets to define mechanisms of thiol-based redox regulation.